The quantification of chemical and biochemical components in colored aqueous fluids, in particular, colored biological fluids such as whole blood and urine and biological fluid derivatives such as serum and plasma, is of ever-increasing importance. Important applications exist in medical diagnosis and treatment and in the quantification of exposure to therapeutic drugs, intoxicants, hazardous chemicals, and the like. In some instances, the amounts of materials being determined are either so minuscule -- in the range of a microgram or less per deciliter or so difficult to precisely determine that the apparatus employed is complicated and useful only to skilled laboratory personnel. In this case, the results are generally not available for some hours or days after sampling. In other instances, there is often an emphasis on the ability of lay operators to perform the test routinely, quickly, and reproducibly outside a laboratory setting with rapid or immediate information display.
One common medical test is the measurement of blood glucose levels by diabetics. Current teaching counsels diabetic patients to measure their blood glucose level from two to seven times a day, depending on the nature and severity of their particular cases. Based on the observed pattern in the measured glucose levels, the patient and physician together make adjustments in diet, exercise, and insulin intake to better manage the disease. Clearly, this information should be available to the patient immediately.
Many blood glucose test methods and test articles are known in the art; these all suffer from a variety of limitations. A new procedure system for the determination of analytes has been shown to overcome these limitations; this procedure system is disclosed and claimed in U.S. Pat. No. 4,935,346 by R. Phillips et al and is assigned to the same assignee as the present application.
The method disclosed and claimed in this patent involves taking a reflectance reading from one surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to another surface and migrates through the matrix to the surface being read. The reagent includes glucose oxidase, which consumes glucose in the sample to produce hydrogen peroxide, which then reacts with a dye couple comprising 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) and 3-dimethylaminobenzoic acid (DMAB) to yield a blue color dye stuff. Reflectance measurements are then made at two separate wavelengths. The concentration of the glucose in blood is determined based on the intensity of the dye color with the aid of a LED spectrophotometer.
The method disclosed and claimed in the above-mentioned patent represents an important step forward in the measurement of blood glucose levels. However, in order to avoid spectral interference with hemoglobin, the glucose measurement is set at 635 nm (in the blue spectral region). This wavelength coincides with the sloping portion of the MBTH-DMAB spectrum, making precise photometric determination difficult without an extensive calibration of the light emitting diode (LED) optics.
Further, the MBTH-DMAB dye couple is very soluble in aqueous media. As the dye forms by the oxidative reaction with hydrogen peroxide, it is prone to migrate away from the reaction zone. Consequently, color intensity gradually decreases with time, thus making the precise endpoint determination of the reaction difficult.
Thus, a need remains in the art to provide a dye couple which produces a blue compound, exhibits a substantially flat absorption in the blue spectral region, and is substantially insoluble in aqueous media upon coupling.